Rb-null macrophages are competent for erythroblast interactions in vivo. (A-C) Immunohistochemical staining for F4/80 on Rb-null fetal liver showing the increased numbers of F4/80⁺ macrophages accumulating in the distal tip of the liver (red arrow, B), and the difference in staining of F4/80 in healthy regions of the liver to the right of the red line (D) compared with dying and ischemic regions of the liver to the left of the red line (C). (D-E) F4/80 immunohistochemistry on intact Rb-null (F) and wild-type (E) fetal liver with F4/80-positive surface area determined by optical densitometry for DAB positivity. (F) Graphic representation of total fetal liver cell number, number of F4/80-positive macrophages, number of TER119-positive erythroblasts, and ratio of TER119-positive erythroblasts to F4/80-positive macrophages in wild-type and Rb-null fetal livers at E12.5 and E13.5. At E12.5, F4/80-positive macrophages represented 13.2% (± 2.7%) and 15.4% (± 0.9%) of total cell number for wild type and Rb null, respectively, and 13.4% (± 4.0%) for wild-type and 12.2% (± 0.4%) for Rb null at E13.5. (G-H) Protein blue-stained E13.5 wild-type (H) and Rb-null (I) fetal livers showing erythroblastic islands in situ (left) with a color-coded key (right) to indicate distinct cell types, including macrophages (M, green), erythroblasts (E, red), hepatocytes (H, brown), endothelial cell (en, turquoise), and liver sinusoids (S). (I-J) Electron microscopy of macrophage-erythroblast contacts in E13.5 wild-type (J) and Rb-null (K) fetal liver. Erythroblasts (Ery), macrophages (Mac), and hepatocytes (Hep) were distinguished on the basis of mitochondrial size and numbers (hepatocytes have larger, more numerous mitochondria), nuclear and cytoplasmic electron density, and smaller cell size (erythroblasts) and larger cell size, cytoplasmic projections, and inclusion bodies (macrophages). See ″Fluorescence microscopy and island counts″ for image acquisition information.